Oral Microbiota Development in the First 60 Months: A Longitudinal Study

Abstract

Childhood is considered crucial in the establishment of future oral microbiota. However, the precise period of oral microbiota development remains unclear. This study aimed to identify the progression of oral microbiota formation in children. We longitudinally investigated the salivary microbiota of 54 children across 13 time points from 1 wk to 60 mo (5 y) old and their parents at 2 time points as a representative sample of the adult microbiota. Using next-generation sequencing, we obtained 10,000 gene sequences of the 16s rRNA V1-V2 region for each sample. The detection rate in children of 110 operational taxonomic units commonly detected in more than 85% of mothers and fathers, defined as the main constituent bacteria, was 25% at 1 wk old, increased to 80% between 6 and 18 mo old, and reached approximately 90% by 36 mo old. Early main constituent bacteria detected at 1 wk old were limited to Streptococcus, Rothia, and Gemella. At 6 to 18 mo old, the detection rates of various main constituent bacteria, including Neisseria, Haemophilus, and Fusobacterium, increased. UniFrac distance analysis showed that the oral microbiota of children approached that of adults at 6 to 18 mo old. In the weighted UniFrac distance index, unlike the unweighted index, there were no significant changes in children between 36 and 60 mo old from adults, and microbiota formation at 60 mo old was sufficiently advanced to be included within the range of adult individual differences. Our findings suggest that the initial 36 mo, particularly the period from 6 to 18 mo old, consists of a time window for oral microbiota maturation. In addition, the development of microbiota during this period may be critical for future oral disease prevention.

Keywords: child; highly prevalent oral bacteria; microbial colonization; microbial consortium; oral bacteria.

Figure 1.

Rate of highly prevalent oral bacteria (HPOB) in the composition of the salivary microbiota of mothers and fathers. “18M” and “36M” indicate samples collected when their children were 18 and 36 mo old, respectively. HPOB, highly prevalent oral bacteria.

Figure 2.

(A) Changes in the average detection rate of highly prevalent oral bacteria (HPOB) in swab-collected saliva and the average number of erupted teeth at each age. The error bars represent the standard deviation, the black line and black circles illustrate shifts in HPOB, and the green dotted line and green squares indicate changes in the number of erupted teeth until 42 mo old. Although the error bars for the number of erupting teeth exceed the range shown in the figure, the upper limit of the figure is 20 teeth because the maximum number of teeth is 20 until the age of 60 mo. (B) The horizontal axis shows the number of days from the time saliva was collected at 6 mo to the day when baby food was started. A negative value indicates that the baby food had not yet been started at the time of saliva collection. The vertical axis shows the detection rate of HPOB at 6 mo old, and the plot colors are coded by the number of teeth that were erupting at the time of collection. Red circles, green squares, and blue triangles indicate 0, 1, and 2 teeth, respectively. HPOB, highly prevalent oral bacteria.

Figure 3.

Line graphs showing the changes in each HPOB on the trend in the rate of children with detected HPOB. The horizontal axis represents the child’s age in months, and the vertical axis displays the detection rate. The legend provides the OTU number and the name of the assigned bacterial species. (A) The diagram is categorized by genus, excluding HPOB assigned to the genus Streptococcus. The assigned genus name is specified in the title of each panel. The genera (Candidatus Saccharibacteria) and (Peptostreptococcaceae) shown in parentheses were not defined in the database, and the defined names of the higher taxa are shown. (B) Due to a substantial number of HPOB assigned to the genus Streptococcus, individual plots are presented for each assigned species to improve visibility. The title of each panel denotes the assigned bacterial species’ name. HPOB, highly prevalent oral bacteria; OTU, operational classification unit.

Figure 4.

(A, B) Shifts in the UniFrac distance of the children’s microbiota collected by swab at each age and adults (both parents) are shown. The Steel–Dwass test P value was used to determine statistical significance. N.S., P > 0.05. (A) Results of the weighted UniFrac distance. (B) Results of the unweighted UniFrac distance. All statistical tests using the unweighted UniFrac index were P < 0.05. (C, D) Results comparing the UniFrac distance ([C] weighted UniFrac distance and [D] unweighted UniFrac distance) between children’s microbiota collected by the mouth-rinsed method at 60 mo old (60M), that between children’s and adults’ microbiota collected by the mouth-rinsed method, and that between adults’ microbiota are shown. The Steel–Dwass test P value was used to determine statistical significance. **P < 0.01.